The objectives of this study are to undertake a clonal analysis of cellular immune response mediated by cytotoxic T cells (CTL) and by natural killer (NK) cells in an autologous human melanoma system and to examine the cellular and molecular basis of cytotoxic unresponsiveness against autologous melanoma cells. We will examine the hypothesis that cytotoxic unresponsiveness against autologous melanoma cells may result from antigen-specific suppression of cytotoxic lymphocytes (CL). To test this hypothesis, we plan to clone autoreactive CTL following in vitro autologous mixed lymphocyte-tumor cell interaction (MLTI) in the presence or absence of third-party helper cells, demonstrate specificity of cytotoxicity of such clones against autologous targets, and then use the clones in cytotoxicity assays to search for putative cellular and/or humoral suppression factor. Four well-characterized autologous melanoma systems, including a system consisting of a melanoma line (VIP) and an autologous in vitro transformed fibroblast line (VIP-F:T), were chosen for an examination of the feasibility of generating autospecific CTL clones. Autologous cytotoxicity could not be detected in any of these systems prior to in vitro sensitization. The VIP system was used extensively as the model system. CL generated against the VIP melanoma cells in MLTI were expanded in interleukin 2 for 2 weeks. The expanded CL were cloned in limiting dilutions. Two phenotypically homogeneous clones (3:1 and E.5) bearing the OKT8 phenotype were obtained. Both clones expressed cytotoxicity selectively only against the sensitizing autologous target VIP. The in vitro transformed autologous fibroblasts (VIP-F:T) were not lysed. Cytotoxicity assays performed with clone E.5 against the VIP target cells in the presence of autologous unfractionated lymphocytes or serum showed no modulation of autoreactivity of clone E.5. In addition, multiple colonies generated in limiting dilutions in the other systems are being analyzed as potential CTL or NK clones. Finally, an NK-like clone has been generated in another system (ML). This clone exhibited an interesting phenotype (negative for OKT3, OKT4, OKT8, HNK-1 and M-1 phenotypes) and showed characteristics of NK cells in cytotoxicity assays. Presently, extensive analysis is under way on this clone. The results thus far have indicated that analysis of cellular immune response against autologous tumor cells might be feasible using autoreactive clones generated by the in vitro cloning technology currently available.